Molecular analysis of muscarinic receptors and regulation mechanism of dilation in the rat iris.

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The amount of mRNAs of muscarinic acetylcholine receptor subtypes, m1, m2, m3, m4 and m5, in the rat iris was determined quasi-quantitatively using RT-PCR methods. The mRNA expression levels of m2 and m3 were higher than those of m1 and m5 in the rat iris as in other smooth muscle tissues. It was characteristic in the rat iris that the m4 mRNA expression level was high. In addition, the cloning of cDNAs encoding m2, m3 and m4 subtypes in the rat iris was performed using RT-PCR methods. Amino acids sequence of m2 subtype in the rat iris included nine differences in comparison with that in the heart, whereas it was 100% identical to genomic one. To examine the mechanisms underlying the parasympathectomy-induced effects in the rat iris smooth muscles, the mRNA expression levels of muscarinic receptor subtypes were determined in iris from ciliaryganglionectomized rats. The results suggested that the parasympathectomy does not markedly affect the mRNA levels. It was also clarified that the intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in the rat iris dilator under the resting conditions is higher than those in the iris sphincter and many other smooth muscles which do not have an inherent tone. Since the muscarinic receptor stimulation resulted in the decrease in [Ca^<2+>]_i in the rat iris dilator, the muscarinic relaxation appeared to be due to the decrease in [Ca^<2+>]_i. In denervated iris dilators, muscarinic receptor stimulation induced neither the decrease in [Ca^<2+>]_i nor the relaxation.